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Cysteine protease inhimbitors

Inactive Publication Date: 2004-10-07
ARAD DORIT +6
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0015] In one aspect, the present invention is a method of use of cysteine protease inhibitors presented herein for the treatment of diseases or disorders affected by cysteine protease activity. The present invention also includes pharmaceutical preparations comprising at least one of the cysteine protease inhibitors presented herein which, when administered in an effective amount, blocks the deleterious effects of infectious diseases or excessive apoptosis. The pharmaceutical preparation of the present invention can be used for the modulation of cysteine protease activity such as the picomavinis 3C-protease and 3C -protease-like proteins, i.e., proteins having similar enzyme activity and active site structure as determined by homology or by x-ray analysis. The pharmaceutical preparation of the present invention can be used for the modulation of cysteine protease activity such as caspases and caspase-like proteins, i.e., proteins having similar enzyme activity and active site structure as determined by homology or by x-ray analysis. The pharmaceutical preparation of the present invention can preferably be used for the modulation of caspase-3 and caspase-3-like proteins, i.e., proteins having similar enzyme activity and active site structure as determined by homology or by x-ray analysis.
[0084] Birch et al have developed a continuous fluorescence assay to determine kinetic parameters and to screen potential HRV14 3C protease inhibitors. The assay consists of a consensus peptide for rhinoviruses connected to a fluorescence donor group (anthranilic acid; Anc) at the N terminal and to an acceptor group (p-NO.sub.2-Phe; Pnp) at the P4 position, both groups flanking the scissile bond (Gln / Gly). The substrate peptide consists of the following sequence: Anc-Thr-Leu-Phe-Gln-Gly-Pro-V-al-Pnp-Lys. There is a linear time dependent increase in fluorescence intensity as the substrate is cleaved, which allows continuous monitoring of the reaction. Multiwell plates containing one inhibitor per well allows for rapid screening by measuring the fluorescence intensity in each well. (Birch et al. 1995. Protein Expression and Purification 6:609-618).

Problems solved by technology

However, a variety of physiological disorders or diseases have been attributed to the presence of excessive or insufficient levels of cysteine proteases.

Method used

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example 2

Caspase-3 Inhibitors

[0091] Representative compounds of the present invention were purchased as part of a combinatorial library from Nanoscale Combinatorial Synthesis, Inc. (NANOSYN.RTM.; Mountain View, Calif.). A number of other compounds were purchased individually from commercial sources (i.e., Compound TestNumbers cpi0116-cpi0135). A few compounds were custom synthesized (i.e., Compound Test Numbers cpi0139-cpi0141).

[0092] To determine the caspase inhibitory activity of these compounds, the in vitro high throughput caspase-3 assay presented herein was utilized. Purified human recombinant caspase-3, fluorescence labeled substrate (Acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin), and known inhibitor (Z-Asp-Glu-Val-Asp-fluoromethyl ketone) were purchased from Sigma. The enzyme reactions were carried out at room temperature in 10 mM PIPES pH=7.4, 2 mM EDTA, 0.1% CHAPS, 5 mM DTT reaction buffer. Each well on the 96 well plate contained the above reaction buffer plus 55 .mu....

example 3

Computational QSAR Prediction of Blood-Brain Permeability

[0095] To assess whether the compounds of the present invention might be useful in treating neurodegenerative diseases, a QSAR (Quantitative Structure-Activity Relationship) model was used to computationally predict the blood-brain barrier permeability for each compound. We developed the QSAR model using the MOE software package from the Chemical Computing Group. A set of 75 compounds with known blood-brain partition coefficients were obtained from the literature (Luco, J. M. 1999. J Chem Inf Comput Sci 39:396-404). A set of 15 descriptors available in this software package were chosen based on a principle component analysis. The QSAR equation was then obtained by linear regression. The resulting QSAR prediction equation reproduced the test set log BB data to an accuracy of RMSE=0.375975 and R.sup.232 0.781358.

[0096] The results of the study are presented in Table IV as log BB values, i.e., the base ten log of the ratio of con...

example 4

Cross-Reactivity

[0097] To evaluate whether inhibition was specific to caspase-3 or applicable to other cysteine proteases or other proteases, inhibitor molecules were assayed for their capacity to inhibit additional non-caspase proteases. Three proteases, TPCK-trypsin, .alpha.-chymotrypsin and papain, were used to test for protease inhibition cross-reactivity. Protease inhibition assays were performed in 96-well flat-bottomed micro-titer plates with the QuantiCleave.TM. protease assay kit (Pierce, Rockford, Ill.) according to the manufacturer's instructions. Briefly, the assay conditions contained 100 .mu.M compound, 2 mg / mL of succinylated-casein and 0.15 mg / mL of the protease. The assay incubated in a final volume of 150 .mu.Ls 0.05 M sodium borate pH 8.5 buffer (NaB-buffer) for 20 minutes at 25.degree. C. After the protease reaction, 50 .mu.Ls of a trinitrobenzenesulfonic acid (TNBSA) solution (1:15, TNBSA:NaB-buffer) was added to each reaction and further incubated for 20 minute...

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Abstract

Compounds having quinone and quinone analogs useful for pharmaceutical preparations have now been found which inhibit cysteine proteases, in particular, caspases and 3C-cysteine proteases. The cysteine protease inhibitors of the present invention can be identified by their mode of action in disrupting the ability of cysteine proteases and, in particular, caspases to cleave a peptide chain. These compounds are useful in inhibiting cysteine protease or cysteine protease-like proteins and for treating infections diseases or physiopathological diseases or disorders attributed to the presence of excessive or insufficient levels of cysteine proteases.

Description

[0001] The present invention relates to the use of certain classes of cysteine protease inhibitors for the treatment of various diseases including infectious diseases and diseases resulting from inappropriate apoptosis.[0002] Cysteine proteases are a major family of peptide-bond-cleaving hydrolases isolated from viruses, bacterial protozoa, plants, mammals and fungi, wherein the thiol group of a cysteine residue serves as a nucleophile in the catalytic process. Normal protein degradation and processing involve a variety of mechanisms which include cysteine proteases. However, a variety of physiological disorders or diseases have been attributed to the presence of excessive or insufficient levels of cysteine proteases.[0003] One family of cysteine proteases, the caspases (i.e., cysteinyl aspartate-specific proteinases), are involved in the conserved biochemical pathway that mediates apoptosis. Apoptosis is one method by which multicellular organisms eliminate unwanted cells. Apoptosi...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61P9/10A61P17/14A61P19/02A61P21/04A61P25/28A61P35/00A61P37/06C07C49/86C07C50/32C07C50/38C07C225/30C07C233/31C07C233/76C07C251/20C07C251/24C07C251/48C07C251/86C07C271/12C07C271/28C07C271/64C07C309/44C07C317/24C07C323/22C07D211/90C07D213/53C07D213/90C07D215/22C07D215/233C07D215/24C07D215/50C07D221/14C07D223/16C07D239/56C07D241/44C07D263/56C07D263/57C07D307/54C07D307/88C07D333/38C07D417/04C07D471/04C07F15/02
CPCC07C49/86C07C50/32C07C50/38C07C225/30C07C233/31C07C233/76C07C251/20C07C251/24C07C251/48C07C251/86C07C271/12C07C271/28C07C271/64C07C309/44C07C317/24C07C323/22C07C2101/14C07C2102/10C07C2103/74C07D211/90C07D213/53C07D213/90C07D215/233C07D215/24C07D215/50C07D221/14C07D223/16C07D239/56C07D241/44C07D263/57C07D307/54C07D307/88C07D333/38C07D417/04C07D471/04C07F15/02A61P9/10A61P17/14A61P19/02A61P21/04A61P25/28A61P35/00A61P37/06C07C2601/14C07C2602/10C07C2603/74
Inventor ARAD, DORITBOLLON, ARTHUR PYOUNG, DAVID CPOLAND, BRADLEY WPEEK, ANDREW SSHAW, BALINVALLURUPALLI, JYOTHI
Owner ARAD DORIT
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